Genome Engineering with CRISPR-Cas9: Birth of a Breakthrough Technology

00:00:07.25 Hi my name is Jennifer Doudna from UC Berkeley 00:00:09.26 and I'm here today to tell you about how we uncovered 00:00:12.26 a new genome engineering technology. 00:00:15.07 This story starts with a bacterial immune system 00:00:20.12 that means understanding how bacteria 00:00:22.14 fight off a viral infection. 00:00:24.26 It turns out that a lot of bacteria 00:00:28.04 have in their chromosome, 00:00:30.09 which is what you are looking at here 00:00:32.15 a sequence of repeats shown in these black diamonds 00:00:36.18 that are interspaced with sequences 00:00:40.19 that are derived from viruses 00:00:43.08 and these have been noticed by microbiologists 00:00:46.20 who were sequencing bacterial genomes but nobody knew 00:00:50.10 what the function of these sequences might be 00:00:53.06 until it was noticed that they tend to also occur 00:00:58.09 with a series of genes that often encode proteins 00:01:03.29 that have homology to enzymes that do interesting things 00:01:09.03 like DNA repair. 00:01:10.12 So it was a hypothesis that this system 00:01:14.04 which came to be called CRISPR 00:01:16.08 which is an acronym for this type of repetitive locus 00:01:19.14 that these CRISPR systems could actually be 00:01:22.29 an acquired immune system in bacteria 00:01:26.00 that might allow sequences to be integrated 00:01:29.06 from viruses and then somehow used later 00:01:32.07 to protect the cell from an infection 00:01:35.26 with that same virus. 00:01:37.05 So this was an interesting hypothesis 00:01:39.11 and we got involved in studying this 00:01:41.18 in the mid 2000's right after the publication 00:01:44.15 of three papers that pointed out 00:01:47.13 the incorporation of viral sequences 00:01:50.00 into these genomic loci. 00:01:52.07 And so what emerged over the next several years 00:01:55.17 was that in fact these CRISPR systems 00:01:58.08 really are acquired immune systems in bacteria 00:02:01.18 so until this point no one knew that bacteria 00:02:04.24 could actually have a way to adapt 00:02:08.08 to viruses that get into the cell 00:02:10.29 but this is a way that they do it 00:02:12.14 and it involves detecting foreign DNA 00:02:15.17 that gets injected like shown in this example 00:02:18.04 from a virus that gets into the cell 00:02:20.07 the CRISPR system allows integration 00:02:26.06 of short pieces of those viral DNA molecules 00:02:29.15 into the CRISPR locus 00:02:31.07 and then in the second step 00:02:33.27 that is shown here as CRISPR RNA biogenesis 00:02:38.20 these CRISPR sequences are actually transcribed 00:02:42.20 in the cell into pieces of RNA 00:02:45.15 that are subsequently used together 00:02:48.23 with proteins encoded by the CAS genes 00:02:52.09 these CRISPR-associated genes 00:02:54.01 to form interfering or interference complexes 00:02:58.12 that can use the information in the form 00:03:01.17 of these RNA molecules to base pair 00:03:04.08 with matching sequences in viral DNA. 00:03:07.18 So a very nifty way that bacteria 00:03:10.12 have come up with to take their invaders 00:03:13.06 and turn the sequence information against them. 00:03:17.00 So in my own laboratory 00:03:20.21 we have been very interested for a long time 00:03:23.09 in understanding how RNA molecules 00:03:26.03 are used to help cells to figure out 00:03:32.00 how to regulate the expression of proteins 00:03:34.03 from the genome. 00:03:35.05 And so this seemed like also a very interesting 00:03:37.27 example of this and 00:03:39.18 we started studying the basic molecular mechanisms 00:03:42.24 by which this pathway operates. 00:03:45.01 And in 2011 I went to a scientific conference 00:03:49.23 and I met a colleague of mine, 00:03:52.13 Emmanuelle Charpentier who is shown in this picture 00:03:56.10 on the far left and Emmanuelle's lab 00:03:58.14 works on microbiology problems and they are 00:04:02.11 particularly interested in bacteria 00:04:04.00 that are human pathogens. 00:04:06.01 She was studying an organism called 00:04:08.03 Streptococcus pyogenes which is a bacterium 00:04:11.15 that can cause very severe infections in humans 00:04:14.25 and what was curious in this bug was that it 00:04:16.25 has a CRISPR system and in that organism 00:04:19.06 there was a single gene encoding a protein 00:04:21.27 known as Cas9 00:04:23.12 that had been shown genetically to be required 00:04:26.09 for function of the CRISPR system 00:04:28.16 in Streptococcus pyogenes, 00:04:30.23 but nobody knew at the time what the function 00:04:33.05 of that protein was. 00:04:34.20 And so we got together and recruited 00:04:37.20 people from our respective research labs 00:04:40.26 to start testing the function of Cas9. 00:04:43.17 So the key people in the project 00:04:45.20 are shown here in the photograph 00:04:48.00 in the center is Martin Jinek 00:04:50.03 who is a postdoctoral associate in my own lab 00:04:52.17 and next to him in the blue shirt 00:04:54.22 is Kryztof Chylinski who was a student 00:04:57.20 in Emmanuelle's lab 00:04:58.26 and so these two guys together with 00:05:00.21 Ines Fonfara who is on the far right, 00:05:02.10 a postdoc with Emmanuelle 00:05:04.01 began doing experiments across the Atlantic 00:05:07.25 and sharing their data. 00:05:10.09 And what they figured out was that 00:05:13.06 Cas9 is actually a fascinating protein 00:05:16.04 that has the ability to interact with DNA 00:05:19.28 and generate a double stranded break 00:05:22.02 in DNA at sequences that match 00:05:25.06 the sequence in a guide RNA 00:05:27.08 and this slide what you are seeing 00:05:29.06 is that the guide RNA 00:05:30.10 and the sequence of the guide in orange 00:05:32.11 that base pairs with one strand 00:05:34.18 of the double helical DNA 00:05:37.11 and very importantly this RNA 00:05:39.28 interacts with a second RNA molecule 00:05:42.09 called tracr that forms a structure 00:05:46.03 that recruits the Cas9 protein 00:05:47.29 so those two RNAs and a single protein 00:05:50.13 in nature are what are required 00:05:52.23 for this protein to recognize 00:05:56.05 what would normally be viral DNAs 00:05:58.11 in the cell and the protein 00:06:01.13 is able to cut these up, 00:06:02.14 literally by breaking up the double helical DNA. 00:06:05.24 And so when we figured this out 00:06:08.14 we thought: wouldn't it be amazing 00:06:10.26 if we could actually generate a simpler system 00:06:13.29 than nature has done 00:06:15.02 by linking together these two RNA molecules 00:06:18.04 to generate a system that would be a single protein 00:06:20.16 and a single guiding RNA. 00:06:22.28 So the idea was to basically take 00:06:25.17 these two RNAs that you see on the far side 00:06:29.29 of the slide and then basically link them together 00:06:33.19 to create what we call 00:06:35.04 a single guide RNA. 00:06:36.22 So Martin Jinek in the lab 00:06:38.25 made that construct 00:06:40.21 and we did a very simple experiment 00:06:44.22 to test whether we truly had 00:06:46.18 a programmable DNA cleaving enzyme 00:06:49.29 and the idea was to generate short single guide RNAs 00:06:54.04 that recognize different sites in a circular DNA molecule 00:06:59.24 that you see here 00:07:00.21 and the guide RNAs were designed 00:07:03.10 to recognize the sequences shown by the red bars 00:07:06.17 in the slide and the experiment was then 00:07:10.16 to take that plasmid, that circular DNA molecule 00:07:13.21 and incubate it with two different restriction (or cutting) enzymes, 00:07:18.27 one called SalI which cuts 00:07:21.23 the DNA sort of upstream at the far end 00:07:25.05 of the DNA in this picture 00:07:26.12 in the grey box, 00:07:27.19 and the second site being directed 00:07:31.00 by the RNA-guided Cas9 00:07:33.13 at these different sites shown in red. 00:07:35.16 And a very simple experiment 00:07:37.19 we did this incubation reaction 00:07:39.26 with plasmid DNA and this is the result 00:07:43.24 and so this is what you are looking at 00:07:45.28 is an agarose gel 00:07:47.29 that allows us to separate 00:07:49.16 the cleaved molecules of DNA 00:07:51.20 and what you can see is that in each of these reaction lanes 00:07:54.22 we get a different sized DNA molecule released 00:07:58.12 from this doubly digested plasmid 00:08:00.16 in which the size of the DNA 00:08:03.29 corresponds to cleavage at the different sites 00:08:06.11 directed by these guide RNA sequences 00:08:08.26 indicated in red 00:08:10.24 so this was a really exciting moment 00:08:12.29 actually a very simple experiment that was 00:08:15.15 kingd of an “A ha!” moment 00:08:17.03 when we said we really have a programmable DNA cutting enzyme 00:08:22.02 and that we can program it with a short piece of RNA 00:08:24.15 to cleave essentially any double stranded DNA sequence 00:08:28.07 so the reason we were so excited 00:08:30.23 about an enzyme that can be programmed 00:08:33.19 to generate double stranded DNA breaks 00:08:36.01 at any sequence is because 00:08:39.00 there was a long standing set of experiments 00:08:42.16 in the scientific community that showed 00:08:45.13 that cells have ways of repairing double stranded DNA breaks 00:08:49.26 that lead to changes 00:08:52.02 in the genomic information in DNA 00:08:55.21 so this is a slide that shows that 00:08:58.20 after a double stranded break is generated 00:09:01.14 by any kind of enzyme that might do this 00:09:04.13 including the Cas9 system 00:09:06.05 those double stranded breaks in a cell 00:09:09.07 are detected and repaired by two types of pathways 00:09:13.15 one on the left that involves 00:09:17.26 non-homologous end joining 00:09:20.07 which the ends of the DNA are chemically ligated 00:09:24.07 back together usually with introduction 00:09:26.18 of a small insertion or deletion 00:09:28.25 at the site of the break 00:09:29.27 and on the right hand side 00:09:32.01 is another way that repair occurs 00:09:34.00 through homology directed repair 00:09:37.22 in which a donor DNA molecule 00:09:39.14 that has sequences that match those 00:09:43.28 flanking the site of of the 00:09:45.12 double stranded break can be integrated 00:09:48.05 into the genome at the site 00:09:50.10 of the break to introduce new genetic information 00:09:54.06 into the genome 00:09:55.15 so this had given many scientists 00:09:59.04 the idea that if there were a tool 00:10:01.04 or a technology that allowed 00:10:03.05 scientists or researchers to introduce 00:10:06.12 double stranded breaks at targeted sites 00:10:09.00 in the DNA of a cell then together 00:10:12.12 with all of the genome sequencing data 00:10:14.21 that are now available we know the 00:10:16.10 whole genetic sequence of a cell 00:10:18.21 and if you knew where a mutation occurred 00:10:21.20 that causes a disease for example 00:10:23.20 you could actually use a technology like this 00:10:26.25 to introduce DNA that would fix a mutation 00:10:31.00 or generate a mutation 00:10:32.23 you might like to study in a research setting 00:10:35.04 so the power of this technology is 00:10:38.28 really the idea that we can now generate 00:10:41.20 these types of double stranded breaks 00:10:43.17 at sites that we choose as scientists 00:10:46.17 by programming Cas9 and then allow 00:10:48.13 the cell to make repairs that introduce 00:10:51.04 genomic changes at sites of these breaks 00:10:54.15 but the challenge was how to generate the breaks 00:10:58.11 in the first place and so a number 00:11:00.09 of different strategies had been produced 00:11:03.24 for doing this in different labs 00:11:05.15 most of them, and I'm going to show 00:11:08.21 two specific examples here 00:11:10.17 one called zinc finger nucleases 00:11:12.25 and the other TAL effector domains 00:11:15.02 these are both programmable ways 00:11:18.08 to generate double stranded breaks in DNA 00:11:20.21 that will rely on protein-based recognition 00:11:23.29 of DNA sequences so these are proteins 00:11:26.06 that are modular, and can be generated 00:11:29.12 in different combinations of modules 00:11:31.22 to recognize different DNA sequences 00:11:34.03 it works as a technology 00:11:37.18 but it requires a lot of protein engineering 00:11:40.24 to do so, and what is really exciting 00:11:43.16 about this CRISPR/Cas9 enzyme 00:11:46.11 is that it is a RNA programmed protein 00:11:49.25 so a single protein can be used for 00:11:52.09 any site of DNA where we 00:11:54.27 would like to generate a break 00:11:56.13 by simply changing the sequence 00:11:58.16 of the guide RNA associated with Cas9 00:12:00.24 so instead of relying on protein-based recognition 00:12:03.06 of DNA we're relying on 00:12:06.04 RNA-based recognition of DNA 00:12:08.26 as shown at the bottom so what this means 00:12:11.03 is that is just a system 00:12:12.18 that is simple enough to use 00:12:15.15 that anybody with basic molecular biology training 00:12:19.05 can take advantage of this system 00:12:20.29 to do genome engineering 00:12:22.20 and so this is a tool that really 00:12:26.06 I think, fills out an essential 00:12:29.03 and previously missing component 00:12:30.24 of what we could call biology's IT toolbox 00:12:33.29 that includes not only the ability 00:12:36.00 to sequence DNA and look 00:12:38.06 at its structure, we know about 00:12:39.24 the double helix since the 1950's 00:12:42.00 and then in the last few decades 00:12:44.18 it's been possible to use enzymes 00:12:46.09 like restriction enzymes 00:12:47.26 and the polymerase chain reaction 00:12:49.09 to isolate and amplify particular segments 00:12:52.19 of DNA and now with Cas9 00:12:55.10 we have a technology that enables 00:12:57.15 facile genome engineering 00:12:59.13 that is available to labs around the world 00:13:03.07 for experiments they might want to do 00:13:05.08 and so this is a summary of the technology 00:13:10.11 of the 2-component system 00:13:11.29 it relies on RNA-DNA base paring 00:13:14.16 for recognition 00:13:15.21 and very importantly because of the way 00:13:18.24 that this system works it 00:13:19.28 is actually quite straight forward 00:13:21.18 to do something called multiplexing 00:13:24.20 which means we can program Cas9 00:13:27.00 with multiple different guide RNAs 00:13:28.23 in the same cell to generate 00:13:30.19 multiple breaks and do things 00:13:32.06 like cut out large segments of a chromosome 00:13:35.01 and simply delete them in one experiment. 00:13:38.25 And so this has led to a real explosion 00:13:42.03 in the field of biology and genetics 00:13:45.19 with many labs around the world 00:13:48.08 adopting this technology 00:13:49.25 for all sorts of very interesting 00:13:51.15 and creative kinds of applications 00:13:53.13 and this is a slide 00:13:54.24 that's actually almost out of date now 00:13:56.11 but just to give you a sense 00:13:57.14 of the way that the field 00:14:00.07 has really taken off 00:14:01.08 so we published our original work on Cas9 00:14:04.10 in 2012 and up until that point 00:14:07.16 there was very little research 00:14:08.18 going on on CRISPR biology anywhere 00:14:11.12 it was a very small field 00:14:12.19 and then you can see that 00:14:13.27 starting in 2013 and extending 00:14:16.09 until now there has been this 00:14:17.21 incredible explosion in publications 00:14:20.16 from labs that are using 00:14:22.09 this as a genome engineering technology 00:14:24.01 so it's been really very exciting for me 00:14:26.25 as a basic scientist to see what started 00:14:29.22 as a fundamental research project 00:14:31.12 turned into a technology that turns out 00:14:34.08 to be very enabling for all sorts 00:14:35.28 of exciting experiments 00:14:37.07 and I just wanted to close by sharing 00:14:40.07 with you a few things 00:14:42.17 that are going on using this technology 00:14:44.17 so of course on the left hand side 00:14:47.13 lots of basic biology that can be done now 00:14:50.02 with the engineering of model organisms 00:14:53.03 and different kinds of cell lines 00:14:55.02 that are cultured in the laboratory 00:14:56.21 to study the behavior of cells 00:14:58.13 but also in biotechnology being able to 00:15:01.23 make targeted changes in plants 00:15:05.15 and various kinds of fungi that could be very 00:15:07.11 useful for different sorts of industrial applications 00:15:09.29 and then of course in biomedicine 00:15:12.14 with lots of interest in the potential 00:15:14.14 to use this technology as a tool 00:15:17.03 for really coming up with novel therapies 00:15:21.06 for human disease I think is something 00:15:23.09 that is very exciting and is really something 00:15:26.05 that is on the horizon already 00:15:27.09 and then this slide just really indicates 00:15:30.20 where I think we're going to see this going 00:15:33.15 in the future with a lot of interesting 00:15:36.20 and creative kinds of directions 00:15:39.03 that are coming along in different labs 00:15:41.02 both in academic research laboratories 00:15:43.19 but also increasingly in commercial labs 00:15:45.29 that are going to enable the use of this 00:15:49.24 technology for all sorts of applications 00:15:52.18 many of which we couldn't even have 00:15:54.10 imagined even two years ago. 00:15:56.05 So very exciting and I want to just acknowledge a great team 00:16:01.10 of people that have been involved in working 00:16:04.22 on the project with me and we've 00:16:06.07 had terrific financial support from various groups 00:16:11.00 as well and it's been a pleasure 00:16:13.00 to share this with you, thank you.

Talk Overview

Jennifer Doudna tells the story of how studying the way bacteria fight viral infection turned into a genomic engineering technology that has transformed molecular biology research. In 2013, Doudna and her colleagues developed the CRISPR-Cas9 gene expression system that, when introduced into animal cells, makes site-specific changes to intact genomes. CRISPR-Cas9 is more precise, more efficient, and less expensive than other genome editing tools and, as a result, has facilitated a wide range of studies that were previously unachievable.

Speaker Bio

Jennifer Doudna is Professor of the Departments of Chemistry and of Molecular and Cell Biology at University of California, Berkeley and an Investigator of the Howard Hughes Medical Institute. Early in her career, she studied the structure and mechanism of ribozymes (enzymatic RNA molecules) and RNA-protein complexes. Now her research focuses on understanding how RNA… Continue Reading

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This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. MCB-1052331.

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